Salmonella typhimurium - sipD (Salmonella invasion protein D)
*Target (protein/gene name):sipD (Salmonella invasion protein D) *NCBI Gene # or RefSeq#:1249312 *Protein ID (NP or XP #) or Wolbachia#:NP_461804.1 , NP_457277.1 *Organism (including strain): species: Salmonella enterica,(subgroup: Salmonella typhi), 2 strains: 98% identical: CT18 (antibiotic resistant) and Ty2 ("normal") Etiologic Risk Group (see link below): RG2
*/Disease Information (sort of like the Intro to your Mini Research Write up): Salmonella typhi is a rod-shaped gram negative bacteria with 2 separate strains: CT18 which contains a plasmid with multiple drug resistance (pHCM1) and TY2 which does not have plasmids and can be affected by antibiotics. Its sole reservoir is in humans, leading to systemic infections and typhoid fever. Once ingested S. typhi passes from the stomach to the intestines and takes residence in the liver or spleen, some of it is expelled as diarrhea. In regions with poor sanitation, it is spread mostly through contaminated water and undercooked food. Salmonella typhi is a food borne pathogen with an ability to change both genotype and phenotype in response to environmental conditions. With the global emergence of multi drug resistant strains, research in the organism has since increased. Current vaccines are developed using the S. typhi TY2 strain and ongoing research is being done to find easier ways to detect typhoid fever (dipstick assay).
Virulence factors (SPI-1- and SPI-2-encoded type III secretion systems) were found during infection. SPI1 (Salmonella pathogenicity island 1) codes for T2SS (type III secretion system) resulting in a suite of proteins that are related to infecting and surviving in the host. SipD in particular is the protein located at the tip of T3SS and is hypothesized to regulate the levels of other proteins (sipA-C among others, sipB in high concentrations is known to cause apoptosis in macrophages).
Link to TDR Targets page (if present):none Link to Gene Database page (NCBI, TCDB) Essentiality of this protein:SipD is one of the essential tip proteins of the T3SS complex that interacts with bile salts that serve as environmental sensors. SipD is required for translocation of effector proteins and is necessary for efficient bacterial internalization and may modulate the secretion of SipA, SipB and SipC (3 other virulence proteins). Studies have been done on both Salmonella and Shingella.
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer: 4-mer Complex of proteins?:yes, 4 identical polypeptide strands arranged into 1 protein Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):research is still being done- most homologous to IpaD protein in Shingella, but also homologous to other T3SS proteins like BipD protein in Burkholderia pseudomallei
*EC#: none assigned, reaction catalyzed is under investigation Link to BRENDA EC# page:none known -- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):none known- (natural substrate is deoxycholate which has aromatic rings- should be able to be detected through spectrophotometric assays) standard gentamicin protection assay (survival/infection assay) quantifies the ability for pathogenic bacteria to invade eukaryotic cells -- link to Sigma (or other company) page for assay (see Sigma links below)(Bio-protocol) -- -or link (or citation) to paper that contains assay information (paper) -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2YM9(SipD from Salmonella typhimurium)
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Figure1.. Crystal structure of the SipD-deoxycholate complex. Deoxycholate (shown as a stick model) binds at the interface of molecules A and B of the asymmetric unit. SipD is colored as follows: the coiled coil (helix α4 and α8), gray; the N-terminal region (helix α1-α3), blue; and the mixed α/β domain, green. The crystal structures of SipD-chenodeoxycholate complex and apo SipD (WT and C244S) are similar to the SipD-deoxycholate crystal and are shown in the Supporting Information.
Current Inhibitors:deoxycholate- impedes host invasion, PRGL Expression Information (has it been expressed in bacterial cells):Transcriptionally regulated by SicA and InvF. Also regulated by InvE. Most commonly expressed in chicken and mice. Purification Method:(SipD) tags include: GST-tag, His-tag, and His-SUMO tag Image of protein (PyMol with features delineated and shown separately): Figure2. PyMol depiction of sipD protein (PDB: 2YM9) from Salmonella typhi shown as cartoon with each chain colored a different color. Each subunit is identical in sequence. (2ym9.pdb)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):sipD has 4 chains that are represented by 1 sequence unique entity (SipD)
*length of your protein in Amino Acids: 346 x 4 = 1,384 residues Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website37393.6 kDa x 4 = 149,574.4 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 38390 (Expasy) TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. Figure3. TMpred graph of 1 of the 4 chains in sipD protein of Salmonella typhi.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
Salmonella typhimurium - sipD (Salmonella invasion protein D)
*Target (protein/gene name): sipD (Salmonella invasion protein D)
*NCBI Gene # or RefSeq#: 1249312
*Protein ID (NP or XP #) or Wolbachia#: NP_461804.1 , NP_457277.1
*Organism (including strain): species: Salmonella enterica,(subgroup: Salmonella typhi), 2 strains: 98% identical: CT18 (antibiotic resistant) and Ty2 ("normal")
Etiologic Risk Group (see link below): RG2
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Salmonella typhi is a rod-shaped gram negative bacteria with 2 separate strains: CT18 which contains a plasmid with multiple drug resistance (pHCM1) and TY2 which does not have plasmids and can be affected by antibiotics. Its sole reservoir is in humans, leading to systemic infections and typhoid fever. Once ingested S. typhi passes from the stomach to the intestines and takes residence in the liver or spleen, some of it is expelled as diarrhea. In regions with poor sanitation, it is spread mostly through contaminated water and undercooked food. Salmonella typhi is a food borne pathogen with an ability to change both genotype and phenotype in response to environmental conditions. With the global emergence of multi drug resistant strains, research in the organism has since increased. Current vaccines are developed using the S. typhi TY2 strain and ongoing research is being done to find easier ways to detect typhoid fever (dipstick assay).
Virulence factors (SPI-1- and SPI-2-encoded type III secretion systems) were found during infection. SPI1 (Salmonella pathogenicity island 1) codes for T2SS (type III secretion system) resulting in a suite of proteins that are related to infecting and surviving in the host. SipD in particular is the protein located at the tip of T3SS and is hypothesized to regulate the levels of other proteins (sipA-C among others, sipB in high concentrations is known to cause apoptosis in macrophages).
Link to TDR Targets page (if present): none
Link to Gene Database page (NCBI, TCDB)
Essentiality of this protein: SipD is one of the essential tip proteins of the T3SS complex that interacts with bile salts that serve as environmental sensors. SipD is required for translocation of effector proteins and is necessary for efficient bacterial internalization and may modulate the secretion of SipA, SipB and SipC (3 other virulence proteins). Studies have been done on both Salmonella and Shingella.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer: 4-mer
Complex of proteins?: yes, 4 identical polypeptide strands arranged into 1 protein
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): research is still being done- most homologous to IpaD protein in Shingella, but also homologous to other T3SS proteins like BipD protein in Burkholderia pseudomallei
*EC#: none assigned, reaction catalyzed is under investigation
Link to BRENDA EC# page: none known
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): none known- (natural substrate is deoxycholate which has aromatic rings- should be able to be detected through spectrophotometric assays)
standard gentamicin protection assay (survival/infection assay) quantifies the ability for pathogenic bacteria to invade eukaryotic cells
-- link to Sigma (or other company) page for assay (see Sigma links below) (Bio-protocol)
-- -or link (or citation) to paper that contains assay information (paper)
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2YM9(SipD from Salmonella typhimurium)
Figure1.. Crystal structure of the SipD-deoxycholate complex. Deoxycholate (shown as a stick model) binds at the interface of molecules A and B of the asymmetric unit. SipD is colored as follows: the coiled coil (helix α4 and α8), gray; the N-terminal region (helix α1-α3), blue; and the mixed α/β domain, green. The crystal structures of SipD-chenodeoxycholate complex and apo SipD (WT and C244S) are similar to the SipD-deoxycholate crystal and are shown in the Supporting Information.
Current Inhibitors: deoxycholate- impedes host invasion, PRGL
Expression Information (has it been expressed in bacterial cells): Transcriptionally regulated by SicA and InvF. Also regulated by InvE. Most commonly expressed in chicken and mice.
Purification Method: (SipD) tags include: GST-tag, His-tag, and His-SUMO tag
Image of protein (PyMol with features delineated and shown separately):
Figure2. PyMol depiction of sipD protein (PDB: 2YM9) from Salmonella typhi shown as cartoon with each chain colored a different color. Each subunit is identical in sequence. (2ym9.pdb)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): sipD has 4 chains that are represented by 1 sequence unique entity
(SipD)
>2YM9:A|PDBID|CHAIN|SEQUENCE
GSHMLNIQNYSASPHPGIVAERPQTPSASEHVETAVVPSTTEHRGTDIISLSQAATKIHQAQQTLQSTPPISEENNDERT
LARQQLTSSLNALAKSGVSLSAEQNENLRSAFSAPTSALFSASPMAQPRTTISDAEIWDMVSQNISAIGDSYLGVYENVV
AVYTDFYQAFSDILSKMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGVKVATEAEARQWLSE
LNLPNSCLKSYGSGYVVTVDLTPLQKMVQDIDGLGAPGKDSKLEMDNAKYQAWQSGFKAQEENMKTTLQTLTQKYSNANS
LYDNLVKVLSSTISSSLETAKSFLQG
*length of your protein in Amino Acids: 346 x 4 = 1,384 residues
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 37393.6 kDa x 4 = 149,574.4 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 38390 (Expasy)
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure3. TMpred graph of 1 of the 4 chains in sipD protein of Salmonella typhi.
*CDS Gene Sequence (paste as text only): (link)
*GC% Content for gene: 41.702128%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**